RNA interference is a method of eliminating gene expression via post-transcriptional gene silencing. RNAi is activated by introducing double-stranded RNA homologous to the target gene transcript. The exogenous RNA is digested in to small interfering RNAs (siRNA), which bind a nuclease complex to form an RNA-induced silencing complex. This complex targets the endogenous gene transcripts by base-pairing and cleaves the mRNA. This methodology is achieved both quickly and easily in both animal and cell-line models. The success of this methodology depends on choosing the proper target sequence within the gene of interest and proper design of the siRNA oliginucleotides. PAGE purification of your oligonucleotides increases the chance of cloning a complete and functional insert. You will need a gene-specific assay to test for suppression of your gene of interest. This assay may be Western blot with an antibody to a protein encoded by your gene, RT-PCR using primers to your gene, Northern blot with a probe to your gene or some other functional assay for the protein encoded by your gene.
Selecting Target Sequences: To identify an optimal target sequence, search for AA dimers within the coding sequence of your gene of interest. Identify 19 nucleotides downstream of the dimer. Don't select sequences within the 5' and 3' untranslated regions and regions near the start codon (w/i 75 bases). Calculate the GC content of the 19-base oligo. It should be between 30-70%. 50% is ideal. Including the AA dimer, check the 21-base oligo for secondary structure and long base runs which interfere with proper annealing. Eliminate these candidates. Compare remaining candidates to a genome database to identify sequences that are specific for the gene of interest and show no significant homology to other genes. We recommend that you test more than one siRNA target sequence for a gene. We can produce multiple siRNAs for you, we recommend testing at least 4 siRNAs per gene. It may be beneficial to choose siRNA targets positioned all along a gene.
Designing Oligonucleotides: Two complementary oligonucleotides (top and bottom strands) are required for each siRNA target site. These should include:
- A 5'-BamH I restriction site overhang on the top strand and a 5'-EcoR I restriction site overhang on the bottom strand.
- A puring (G or A) residue located just downstream of the BamH I site on the top strand
- The 19-base oligo sense sequence of the siRNA target site
- A 7-9 nucleotide hairpin loop sequence (5'-TTCAAGAGA-3' is good).
- The 19-base oligo antisense sequence of the siRNA target site
- a RNA pol III terminator sequence consisting of a 5-6 nucleotide poly(T) tract
- Optional (recommended) a unique restriction site immediately downstream of the terminator sequence for restriction digest analysis to confirm the presence of the cloned insert.
To initiate the service:
We encourage a brief meeting between the investigator and a VDL member to identify the best methodology for constructing their virus. At this stage several options are available based on promoter preference and transgene size. The investigator will then be provided an appropriate adenovirus shuttle plasmid by the VDL.
Once the investigator has cloned the transgene of interest into the provided plasmid, return it to us with the appropriate forms and other requested information.